Usually, the search for an analyte of interest and for the concentration thereof in a whole blood sample likely to contain said analyte of interest comprises, first, separating the plasma from the red cells, particularly by centrifugation, and then measuring the analyte concentration in the plasma.
The “whole blood” corresponds to a biological sample comprising blood with all its components and thus, in particular, the plasma and the red cells. It may for example be blood sampled from a man or an animal without any transformation, or the same blood to which adjuvants have been added, for example, anticoagulants.
Among concentration measurement methods, techniques are known which comprise modifying a measurable property, for example, an optical, electrical, chemical, pH, or enzyme property, according to the quantity of analytes in a blood sample, especially so-called direct, indirect, and competitive ELISA (“Enzyme-Linked ImmunoSorbent Assay”), ELFA (“Enzyme-Linked Fluorescence Assay”), and immunocapture techniques.
FIG. 1 schematically illustrates the main steps of a direct ELISA measurement, also called “sandwich” ELISA since it involves two partners for binding to the analyte, where, for example, the analyte is an antigen and the two binding partners are antibodies each comprising a site, or epitope, different from each other and each capable of binding to said antigen provided with a complementary site. The ELISA measurement comprises, in its simplest version:                coating the surface of a solid support, such as a well, for example, with a layer of a first binding partner of the analyte which is desired to be detected or the concentration of which is desired to be measured, where the first binding partner exhibits a site complementary to the analyte (FIG. 1A);        pouring the blood sample, such as plasma originating from the centrifugation of a whole blood sample, into the well (FIG. 1B) so that the analytes present in the plasma conjugate with the first binding partners fixed to the well wall (FIG. 1C);        performing a first washing of the well to remove the elements of no interest for the ongoing analysis, for example, antibodies and antigens which are not searched for (FIG. 1D);        pouring into the well second binding partners for the analyte, each provided (i) with a site, complementary to one of the free sites of the analytes immobilized by means of the first binding partners and different from the complementary site of the first binding partner, and (ii) with a component having an enzyme function capable of catalyzing a hydrolysis of a substrate which changes color according to the quantity of catalyzed hydrogen (FIG. 1E). Such second binding partners containing an enzyme function are called “conjugates”;        performing a second washing of the well to remove excess conjugates (FIG. 1F);        adding substrate degradable by the enzyme function of the conjugates into the well and measuring the chrominance or optical density of the medium contained in the well by spectrometry (FIG. 1G).        
Since chrominance directly depends on the quantity or amount of analytes immobilized by the first binding partners fixed to the wall of the solid support, the measurement of this optical property thus is an indirect measurement of the total quantity of analytes present in the plasma sample, and accordingly, knowing the volume of the sample, of the analyte concentration therein. The measured chrominance is then transformed, by means of a predetermined mathematical model, into an amount and/or concentration value. “Amount” thus means a quantity of analytes in a sample. “Concentration” means an amount divided by the volume of the sample whereon the measurement is performed.
“Competitive” ELISA involves a single binding partner for the analyte, which exhibits a site complementary to one of the sites of the analyte, as well as a compound which competes with the analyte to be assayed. Once of these two elements then has an enzyme function capable of catalyzing a hydrolysis of a substrate which changes color according to the quantity of catalyzed hydrolysis. The amount and thus the concentration are thus inversely proportional to the read chrominance.
ELFA measurements are similar to ELISA measurements, but for the fact that the substrate catalyzed by the enzyme function generates fluorescence measured, for example, by a fluorometer.
All these measurement techniques provide elements only fixing the analytes having their concentration desired to be measured. Although only the latter are specifically fixed, it can however be observed that the nature of the sample substantially influences the amount measurement, especially when the reaction time is decreased due to diagnosis-related time constraints. Thus, when the measurement is directly performed on a whole blood sample, the measured amount is smaller than that which is directly measured on a plasma sample, particularly due to the presence of haematocrit, which has a matrix effect.
As known per se, the haematocrit level, or haematocrit, corresponds to the relative volume occupied by red cells relative to the whole blood volume. Certain manufacturers of immunoassay instrument using, as a sample for the diagnosis, a whole blood sample instead of a plasma sample, have provided correcting the analyte amount measured in a whole blood sample according to the following formula:
                              D          p                =                              D            ST                                1            -                                          D                H                            100                                                          (        1        )            where Dp is the corrected analyte amount, DST is the analyte amount directly measured on a whole blood sample, DH is a measurement of the haematocrit level of the whole blood sample. It should be noted that relation (1) may also apply with the measured and corrected analyte concentrations instead of the corresponding amounts. However, such a correction, which is similar to a simple rule of three, does not provide good results.
To date, the amount of an analyte, and accordingly the concentration thereof, is thus mainly determined on plasma, this measurement being the only one considered as reliable and reproducible. Further, such a measurement has the advantage of being independent from the haematocrit level, which is variable from one subject to the other.
Now, the obtaining of plasma requires a prior centrifugation step, and thus time and specific equipment. Not only may time be a crucial parameter in the case of medical diagnosis, especially when there a threats on the subject's life, but this further assumes a large quantity of centrifuges in a laboratory in charge of a large number of analyses.